Imaging Retinal Melanin: a Review of Current Technologies Maryse Lapierre-Landry1,2,6 , Joseph Carroll3,4 and Melissa C

Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 https://doi.org/10.1186/s13036-018-0124-5

REVIEW Open Access Imaging retinal melanin: a review of current technologies Maryse Lapierre-Landry1,2,6 , Joseph Carroll3,4 and Melissa C. Skala1,5*

Abstract The retinal pigment epithelium (RPE) is essential to the health of the retina and the proper functioning of the photoreceptors. The RPE is rich in melanosomes, which contain the pigment melanin. Changes in RPE pigmentation are seen with normal aging and in diseases such as albinism and age-related macular degeneration. However, most techniques used to this day to detect and quantify ocular melanin are performed ex vivo and are destructive to the tissue. There is a need for in vivo imaging of melanin both at the clinical and pre-clinical level to study how pigmentation changes can inform disease progression. In this manuscript, we review in vivo imaging techniques such as fundus photography, fundus reflectometry, near-infrared autofluorescence imaging, photoacoustic imaging, and functional optical coherence tomography that specifically detect melanin in the retina. These methods use different contrast mechanisms to detect melanin and provide images with different resolutions and field-of-views, making them complementary to each other. Keywords: Retina, Retinal pigment epithelium, Choroid, Fundus reflectometry, Photoacoustic, Autofluorescence, Near- infrared, Optical coherence tomography, Photothermal, Polarization

Background intact living eye. To further study the RPE, new imaging Melanin is naturally present in the eye within the choroid, techniques have been developed to specifically detect and iris, and retinal pigment epithelium (RPE), a single layer of quantify melanin at the clinical and pre-clinical levels in epithelial cells located posterior to the photoreceptors in patients and animal models. the retina. The RPE plays an important role in the overall Eye imaging has multiple roles, both to improve patient health of the retina, transporting nutrients from the blood care and to perform basic research. Clinical imaging is used vessels in the choriocapillaris to the photoreceptors, and in patients to screen and diagnose eye conditions, plan and disposing of retinal waste and metabolic end products [1]. monitor ocular surgeries and evaluate treatment response An interruption in these functions can lead to degener- [6, 7]. In animal models, non-invasive imaging methods ation of the retina, loss of the photoreceptors and eventu- enable observation of how different ocular structures inter- ally blindness. The melanin in the RPE is thought to play act with each other in a living system. Disease progression a protective role, absorbing excess light from the photore- can be studied over time in the same animal, which can ceptors and protecting the retina from light-generated lead to the identification of new disease markers. Alterna- oxygen reactive species [2–4]. However, melanin in the tively, new drugs can be dynamically evaluated, which could RPE does not regenerate, and the damage accumulated accelerate clinical translation. Fundus photography, scan- over time from light exposure could affect the overall ning laser ophthalmoscopy (SLO) and optical coherence health of the RPE [2, 5]. In the past, most methods avail- tomography (OCT) are all non-invasive imaging techniques able to researchers to study melanin in the RPE were that are part of the toolset for clinicians and researchers to destructive to the tissue and labor intensive, which has led image the eye. These techniques could be adapted to image to a limited understanding of the role of melanin in the melanin in the living eye and improve our knowledge of the RPE. * Correspondence: [email protected] Changes in retinal pigmentation normally happen with 1Morgridge Institute for Research, Madison, WI, USA 5Department of Biomedical Engineering, University of Wisconsin Madison, aging [8] and are present in many ocular diseases. Albinism, Madison, WI, USA for example, is characterized by various degrees of ocular Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 2 of 13

hypopigmentation and is associated with low visual acuity Quantifying melanin ex vivo and other visual abnormalities [2]. Retinitis pigmentosa, Multiple methods have been developed to quantify mel- another example, is a group of genetic disorders that cause anin in cells or in ex vivo tissue samples. In early studies progressive visual loss and includes both photoreceptor of the RPE, changes in pigmentation were observed quali- degeneration and RPE cells loss [9]. Finally, age-related tatively [17, 18]orquantitatively[19] by counting melano- macular degeneration (AMD) is the most important cause somes on high resolution micrographs. To accelerate the of vision loss in adults above 65 years old in the US and in- process, melanin is now quantified using chemical degrad- volves dysfunction of the RPE and changes in pigmentation ation of the sample followed by high-performance liquid [10]. At early stages of the disease, AMD is usually charac- chromatography (HPLC) [20]. Electron spin resonance terized by changes in pigmentation and the presence of spectroscopy (ESR) has also been used to quantify melanin drusen. At later stages, “dry” AMD is characterized by re- and characterize the different types of melanin pigments gions of atrophy of the RPE and photoreceptors, while in [5, 21, 22]. ESR spectroscopy measures the magnetic field “wet” AMD neovascular lesions invade the retina from the strengths at which electrons in a sample can change their choroid and lead to vascular leakage, scaring and central spin magnetic moment (from parallel to anti-parallel) by vision loss [11]. In dry AMD, hyperpigmentation in the absorbing the energy from a microwave source of fixed RPE (potentially from dysfunction in the RPE cells) followed frequency. The resulting spectrum of energy absorption as by hypopigmentation (from the loss of RPE cells) could a function of magnetic field strength is specific to a given appear before dysfunction in the photoreceptors or chemical compound and can be used to differentiate choriocapillaris and could be predictive for the progres- pigments. Melanin can also be quantified in terms of light sion of the disease [11]. In wet AMD, it is possible that absorption. Absorbance of solubilized melanin at a spe- loss of the choriocapillaris causes the RPE cells to be- cific wavelength measured with a spectrophotometer is come hypoxic and to produce angiogenic substances, another technique used to quantify melanin in ex vivo resulting in the formation of neovascular lesions [11]. samples [5, 23–25]. Light transmission measurements can To this day, there is no cure for AMD and vision loss also provide a measure of melanin concentration in tissue cannot be reversed, although anti-VEGF treatment can slices [26]. Ex vivo methods provide a highly specific and slow down or stop disease progression [12–14]. quantitative measurement of melanin and are used to Clinical imaging in the eye is already used to facilitate study melanin production, distribution and degradation as diagnosis, evaluate treatment response and reduce the a function of age and diseases. However, these methods need for repeated treatment in AMD [15, 16]. However, cannot be used in live animal models to monitor diseases changes in pigmentations are still difficult to quantify over time or test new treatments, and they cannot be since many non-invasive measurements are highly translated to the clinic for use in patients. As such, in vivo dependent on the optical properties of the eye and on techniques that can detect melanin have been a focus of the imaging parameters used. As a result, there are cur- many researchers. rently no standard in vivo techniques to quantify melanin levels in the eye. Fundus photography and fundus reflectometry The aim of this manuscript is to explore the different Fundus photography is a commonly used clinical imaging ways melanin can be imaged in the living eye. It is be- modality that produces a two-dimensional, en face color lieved that light damage accumulated over time reduces image of the retina where the optic nerve head, macula the melanin’s ability to protect the retina. Imaging and and major blood vessels can be seen. Most modern quantifying melanin in the eye could provide informa- table-top fundus systems have a field-of-view of ~ 45° and tion about the overall health of the RPE and of neighbor- do not require pupil dilation [27]. Fundus images can be ing structures. As a result, melanin imaging could play a recorded on 35 mm-film or with a digital camera [7]. The role in creating and evaluating new treatments in animal basic components of a fundus system are a white light models or diagnosing ocular diseases before irreversible source to illuminate the retina, a central obscuration in vision loss. The following key technologies enable non- the illumination path (annular aperture), an objective lens invasive detection of melanin in the eye at the clinical to form an image using the reflected light from the retina, and pre-clinical level and will be reviewed in this a zoom lens to correct for the patient’s refractive error, manuscript: fundus photography, fundus reflectometry, and a camera to detect the image [28]. This results in near-infrared autofluorescence imaging (NIR-AF), photo- an annular illumination pattern at the pupil, a circular acoustic imaging (PA), optical coherence tomography illumination pattern at the retina and a circular image (OCT), polarization-sensitive OCT (PS-OCT) and photo- detected at the camera. The annular illumination pat- thermal OCT (PT-OCT). A brief summary of existing ex tern at the pupil reduces the back reflection from the vivo methods to quantify melanin in samples is also pre- cornea and allow for a better detection of the reflected sented to provide context. light from the retina. The illumination and collection Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 3 of 13

paths can be combined with a beam splitter, or a mirror density between the eyes of a patient [38]. In another with a central hole to deflect the illumination path study by the same group, fundus reflectometry was used while transmitting the collected light [28]. to image melanin in patients with age-related maculo- Researchers and clinicians can visually assess changes pathy(ARM)butdidnotdetectdifferencesinmel- in pigmentation based on the color of the retina as seen anin optical density between healthy patients and on fundus images. For example, multiple manual grading patients with ARM, or between patients with different systems are used to evaluate fundus images in patients stages of ARM [32]. with AMD and the presence of hypopigmentation or Fundus reflectometry is thus providing quantitative infor- hyperpigmentation is evaluated as part of the overall assess- mation about melanin distribution. This is an improvement ment [29]. Additionally, adaptive optics has been used to over fundus photography where pigmentation changes can correct light aberrations in the eye, effectively improving only be interpreted qualitatively.However,fundusreflect- the lateral resolution of fundus photography, and providing ometry requires complex models to determine how the images of pigment migration over time in “dry” AMD [30]. light entering the eye was scattered and absorbed by the However, this method of evaluating fundus images cannot different tissue layers of the eye. This can lead to widely differentiate between melanin contained in the RPE or the varying results, including non-physical values of melanin choroid, nor is it quantitative. To collect quantitative infor- optical density when layer thicknesses are not estimated mation from the fundus image, fundus reflectometry was correctly [33]. Additionally, while some models can pro- developed. duce 2D images of melanin distribution [37 ], most fundus Fundus reflectometry can be performed with a retinal reflectometry techniques do not produce an image, which densitometer, an instrument composed of a light source, renders data interpretation more difficult and does not some filters to change the wavelength of the light enter- account for heterogenous distributions of melanin. As a ing the eye and a detector such as a photomultiplier, result, fundus reflectometry has not yet become a standard capable of quantifying the light exiting the eye [31]. imaging technique in the clinic and has not been used ex- When performing fundus reflectometry using this tech- tensively to study different diseases of the eye involving nique, a high intensity white light is first sent to the eye melanin. In conclusion, fundus reflectometry can obtain to bleach the retina. A lower intensity light of a specific quantitative measurements of the melanin optical density, wavelength (e.g. 500 nm) is then sent to measure the but the complex models required for quantification make presence of a pigment such as melanin [31, 32]. The light this technology difficult to implement in practice. reflecting from the retina is then quantified as it is reach- ing the detector over time. In other instruments, a white Near-infrared autofluorescence imaging (NIR-AF) light source is used to illuminate the retina and a spec- An alternative to fundus photography is scanning laser oph- trometer is used at the detector to measure the reflected thalmoscopy (SLO) [39], which has enabled near-infrared light at multiple wavelengths [33]. Different theoretical autofluorescence imaging of the eye (NIR-AF). Like fundus models describing how incoming light would be reflected photography, SLO produces two-dimensional en face images or absorbed by the different tissue layers of the retina can of the retina. However, a pinhole can be used to selectively then be fitted to the recorded light, and properties such as collect light from a specific layer of the retina (~ 300 μm the optical density of melanin can be calculated [34]. axial resolution [40]), which is not possible using a fundus Fundus reflectometry studies have found different op- camera [41]. Instead of a white light source, SLO uses a laser tical density values for choroidal melanin in healthy eyes source focused onto a point and raster-scanned across the based on different models [35, 36]. Recently, Hammer et retina to build an image. This enables a small portion of the al. used the adding-doubling approach, a technique used eye’s pupil to be used for illumination, while the rest of the to simulate light distribution in a multi-layered tissue pupil is used for light collection [41]. In comparison, fundus based on the reflection and transmission properties of a photography requires most of the pupil to be used for illu- thin homogeneous tissue layer, to obtain relative concen- mination (annular illumination pattern) with only the center trations of melanin in the RPE and choroid [33]. Bone et of the pupil used for collection. As a result, SLO can be per- al. used a model based on the absorption of four compo- formed with illumination powers much lower than those re- nents (macular pigments, cones and rods, and melanin) quired for fundus photography [39] and SLO is sensitive to at four different wavelength to obtain 2D images of the lower levels of emitted light than fundus photography, enab- fundus (see Fig.1) showing the relative optical density of ling autofluorescence imaging of the eye [42]. Two endogen- melanin [37]. Kanis et al. compared the optical density ous fluorophores are most commonly imaged with SLO: of melanin from the right and left eye of patients and lipofuscin and melanin [43, 44]. In most commercial and found a strong interocular correlation in healthy eyes clinical SLO systems, the choice of excitation and emission [38]. This could open the door to diagnostic tests that wavelengths for fluorescence imaging is often dictated by evaluate large differences between melanin optical the wavelengths used to image two exogenous fluorophores Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 4 of 13

Fig. 1 Pigment distribution obtained using four-wavelengths fundus reflectometry. Relative optical density at the fundus of (a) macular pigment obtained at 460 nm, (b) cone photopigment at 550 nm, (c) rod photopigment at 505 nm, and (d) melanin at 460 nm. Reprinted from [37] with permission from Elsevier that are commonly used in the clinic to perform angiog- subjects and patients with AMD or other retinal diseases raphy: fluorescein and indocyanine green. However, these with NIR-AF, and determined that melanin in the RPE emission and excitation wavelengths are appropriate for and choroid was a likely candidate for the source of the lipofuscin (excitation: 488 nm, emission: > 500 nm, similar near-infrared autofluorescence signal [45]. Finally, Gibbs to fluorescein) and melanin imaging (excitation: 787 nm, et al. demonstrated that the autofluorescence signal was emission: > 800 nm, similar to indocyanine green) [40, 45]. specific to the melanosomes from the RPE and choroid SLO thus enables qualitative imaging of the melanin and its by isolating them ex vivo [49]. distribution throughout the RPE. NIR-AF was performed to detect melanin in patients and The near-infrared autofluorescence signal of melanin study diseases such as AMD [45, 48, 50–52](seeFig.2), in the retina was first reported, to our knowledge, by idiopathic choroidal neovascularization [53], chloroquine Piccolino et al. [46] in 1996 in a study that recorded retinopathy [54], various inherited retinal diseases [55], near-infrared fluorescence before indocyanine green in- ABCA4-associated retinal degenerations [56–58], retinitis jection using fundus photography. At the time it was un- pigmentosa [9, 59, 60], Usher syndromes [49, 61], Best vitel- clear what the source of the fluorescence signal was, and liform macular dystrophy [62], diabetic macular edema the authors hypothesized that it could be a combination [63], central serous chorioretinopathy [64, 65], and torpedo of melanin, lipofuscin, and porphyrins. Later, Huang et maculopathy [66]. NIR-AF has multiple advantages as a al. confirmed that melanin in the skin and synthetic mel- melanin imaging technique: it offers a large imaging field- anin produce fluorescence emission following near-infra- of-view, does not require exogenous contrast agents, is safe red excitation [47]. Weinberger et al. confirmed the and comfortable for the patient, can be performed using results from Piccolino et al. in the eye using an SLO sys- commercially available equipment, and produces images tem and further supported the hypothesis that the NIR that are easy to interpret by researchers and clinicians. fluorescence signal is caused by autofluorescence of However, NIR-AF does not have the axial resolution to pro- melanin and not simply light reflected from the fundus duce three-dimensional images of the melanin distribution (i.e. pseudofluorescence) [48]. Further evidence was and it is likely that melanin from the RPE and choroid are provided by Keilhauer and Delori who imaged normal both contributing to the NIR-AF signal. Additionally, the Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 5 of 13

Fig. 2 Geographic atrophy (GA) in the foveal region due to age-related macular degeneration (AMD) imaged with (a) short-wavelength autofluorescence (SW-AF) to detect lipofuscin, and (b) near-infrared autofluorescence (NIR-AF) to detect melanin. Areas of hypo-fluorescence (c, d) corresponds to GA. Larger areas of hypo-fluorescence are detected with (c) SW-AF compared to (d) NIR-AF, which may indicate that SW-AF overestimates areas affected by GA in the fovea. Reproduced from [50] with permission from BMJ Publishing Group Ltd. interpretation of the NIR-AF is mostly qualitative since the FLIO particularly complementary to NIR-AF. The fluores- fluorescence intensity is highly dependent on imaging con- cence lifetime of melanin has been recorded in hair samples ditions. The NIR-AF signal can thus be quantified within [71]. However, the fluorescence lifetime signal obtained one eye [45, 63] but it has been difficult to directly correlate fromtheretinaincludescontributions not only from mel- theNIR-AFsignaltoanabsolutemeasureofmelanincon- anin but also from multiple fluorophores such as lipofuscin centration that would be valid across multiple eyes. How- and macular pigments [70, 72, 73], and further studies are ever, quantitative autofluorescence has been performed in needed to isolate the lifetime signal of retinal melanin from the eye to quantify lipofuscin in short-wavelength autofluo- other fluorophores in vivo. rescence (SW-AF) images with the use of an internal fluor- escent reference [67–69], which is encouraging for future Photoacoustic imaging (PA) quantitative autofluorescence measurements of melanin in Photoacoustic imaging (PA) is an ultrasound-based mo- the eye. In conclusion NIR-AF is easily performed using dality which can detect optical absorbers such as blood commercially available instruments and has been used to and melanin in the eye [74]. PA uses a pulsed-laser and study multiple human diseases. However, RPE melanin can- an ultrasound transducer to detect absorbers in tissue. not be separated from choroidal melanin and further re- The laser light is absorbed by the contrast agent (e.g. search is needed to obtain quantitative NIR-AF results. melanin), which creates heat, rapid tissue expansion and Fluorescence lifetime imaging ophthalmoscopy (FLIO) an ultrasonic wave via the photoacoustic effect [75]. [70] is a technique similar to NIR-AF that not only mea- Such wave is detected by an ultrasound transducer sures the autofluorescence signal from fluorophores in coupled to the eye. Two types of information about the the retina, but also the time it takes for fluorescence to sample can then be obtained from the ultrasonic wave. be emitted following excitation (i.e. fluorescence lifetime). First, a one-dimensional signal of absorption as a func- The fluorescence lifetime of a fluorophore such as melanin tion of depth into the eye can be computed. The pulsed is highly dependent on the microenvironment but not laser is then scanned across the sample to create two- or dependent on fluorophore concentration, thus making three-dimensional images of the absorbers within the Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 6 of 13

sample. Second, the amplitude of the signal can be cor- the choroid and RPE were then performed in ex vivo sam- related to the absorption coefficient of the sample, and ples using a calibration curve obtained in phantoms. thus can serve as a measurement of the concentration of PA imaging can provide volumetric images of ocular absorber (e.g. melanin) within the sample. melanin, which was not possible using fundus reflectom- As a first demonstration, Silverman et al. acquired PA etry or NIR-AF fundus imaging. The increased axial reso- images of melanin in the iris in excised porcine eyes lution also allows for a more localized signal collection, [76]. In the first in vivo demonstration, Jiao et al. inte- and possibly for independent measurements of RPE and grated PA into an OCT system to collect photoacoustic choroid melanin. PA imaging also relies on simpler light images of the blood and melanin in the healthy rat retina absorption and propagation models than fundus reflect- with a 23 μm axial resolution [77]. This system used a ometry, which may lead to more accurate measurements needle transducer in contact with the eyelid to detect of melanin concentration. However, PA imaging has been the ultrasound signal. Multiple follow-up studies have demonstrated in few animal eye models and has yet to be been produced by the same group. Zhang et al. added demonstrated in the human eye. Additionally, no eye dis- short-wavelength autofluorescence imaging to the PA ease models have been explored using PA, thus it is un- system to detect lipofuscin in addition to melanin, first clear how the information provided by PA imaging will be in retinal tissue [78], then in vivo in pigmented and albino used by eye researchers and clinicians in the future. In rats [79]. Song et al. built upon this work and developed a conclusion, PA imaging provides a quantitative measure- multimodal system that includes PA, SLO, OCT and fluor- ment of melanin absorption and has the potential to sep- escein angiography to image the eye [80]. The resulting arate signal from the RPE and the choroid. However, the system was able to simultaneously image tissue structure, technique has yet to be performed in the human eye. retinal and choroidal blood vessels and melanin from the RPE and choroid in vivo in the retina of albino and pig- Optical coherence tomography (OCT) mented rats [80]. This system was also adapted to image OCT provides three-dimensional, high resolution images melanin in the mouse eye in Song et al. [81]. Previous PA of the different tissue structures of the eye over a large systems by this group had used visible light (532 nm) to field-of-view. First commercialized in 1996, OCT is now excite and detect ocular melanin, however, near-infrared a standard imaging technique both for pre-clinical and light is less damaging to the eye than visible light. Liu et clinical eye imaging [88–90]. OCT uses low-coherence al. thus demonstrated in vivo melanin imaging in rats interferometry to measure the echo time delay and in- using a near-infrared laser (1064 nm) for PA excitation tensity of the backscattered light as it penetrates tissue. [82]. Liu et al. also combined a PA system to a fundus Light is sent into a Michelson interferometer composed camera, which could visualize the position of the PA laser of a beam splitter, a sample arm (ending at the sample, onto the retina and accelerate the alignment procedure in this case the retina) and a reference arm (ending with when imaging melanin in rats [83]. Liu et al. were the first a reflective surface). A Fourier Transform of the result- to perform in vivo optical coherence photoacoustic mi- ing interferogram is used to obtain the OCT signal as a croscopy (PA and OCT combined using the same 800 nm function of depth. The processed OCT signal is thus a wideband light source) in the rat eye, which lead to complex signal where both the signal magnitude and perfectly co-registered images of the tissue structure and phase vary as a function of depth. A single OCT scan melanin distribution (see Fig. 3)[84]. (A-scan) is a one-dimensional measure of sample reflect- Images acquired up to this point had been qualitative ivity as a function of depth. Two- and three-dimensional and suffered from low axial resolution. PA has the po- images can be acquired by raster-scanning the OCT tential to provide a quantitative reading of melanin con- beam over the sample. Typical OCT lateral resolution centration in the eye, similar to previous work imaging falls between 1.5 μm and 9 μm, dependent on the object- cutaneous melanin [85]. Shu et al. performed a Monte ive used and the imaging source wavelength. The axial Carlo simulation to understand light absorption in the resolution is determined by the imaging source wave- retina and evaluate the potential of PA imaging for length and bandwidth, where, up to a point, small wave- quantitative imaging of melanin in the eye [86]. This lengths and large bandwidth lead to better resolution. model used blood absorption as a reference point for Ophthalmic OCT systems will often be centered around calibration. However, to specifically quantify RPE melanin 850-860 nm with a 50 to 100 nm bandwidth, resulting in and separate it from choroidal melanin, a higher axial axial resolutions between 3 μmand6μm[91]. With such resolution was necessary. Shu et al. used a micro-ring res- contrast mechanism and high axial resolution, different onator detector to increase the axial resolution of their PA tissue layers such as the nerve fiber layer, photoreceptors, system (< 10 μm) and obtained images where the RPE and and RPE can be distinguished on OCT images [92]. choroid can be distinguished in ex vivo porcine and hu- Changes in melanin content are visualized as a change in man samples [87]. Quantitative melanin measurements of RPE reflectivity on OCT images. Wilk et al. have analyzed Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 7 of 13

Fig. 3 Optical coherence photoacoustic microscopy acquired in vivo in the rat eye. Top: OCT cross-sectional image showing the retinal tissue layers. Bottom: Co-registered photoacoustic image showing melanin in the RPE and choroid. Red arrow indicates retinal blood vessel. Scale bar: 100 μm. Reprinted from [84]. Copyright Optical Society of America these changes in OCT signal by comparing images obtained volunteer [98]. Follow-up studies by different groups in wild-type and albino zebrafish, and by imaging patients later confirmed that the polarization-scrambling layer with albinism [93]. Zhang et al. have also observed a change was likely the RPE. This conclusion was made by compar- in intensity of the OCT signal in the RPE with dark adapta- ing PS-OCT images obtained in healthy patients and im- tion in frogs [94].However,themainsourceofcontraston ages obtained in patients with RPE detachment, RPE tear, OCT images is tissue backscattering, which provides lim- RPE atrophy, drusen or choroidal neovascular membrane ited functional information and low specificity when im- [99–101]. Baumann et al. used melanin phantoms to deter- aging melanin. Techniques such as polarization-sensitive minethesourceofthePS-OCTsignalwithintheRPE and photothermal OCT have been developed to add func- and observed that the degree of polarization uniformity tional contrast to OCT and can be used to specifically de- (DOPU) is correlated with melanin concentration tect melanin. [102], a result later confirmed in rats [103]. However, Polarization-sensitive OCT (PS-OCT) provides infor- this relationship was strongly dependent on the scatter- mation about the birefringence of a sample and has been ing properties of the sample, i.e. the size and shape of used to image the cornea and retina [95, 96]. To perform the melanin granules [102]. PS-OCT was also per- PS-OCT, incoming OCT light must be circularly polar- formed in pigmented rats and mice [104], albino rats ized. After passing through the sample, the outgoing [103–105], and patients with ocular albinism [102, 106], light then maintains an arbitrary ellipsoid polarization which confirmed the specificity of the PS-OCT signal to pattern determined by the composition of the sample melanin. PS-OCT has been used to segment the RPE from [97]. From there, individual detectors are used to meas- 2D or 3D OCT data sets in healthy eyes [107] and in pa- ure the vertical and horizontal components of the polar- tients affected by AMD [108–111], RPE detachment [111] ized light. Different algorithms are used to extract the and pseudovitelliform dystrophies [108], and to compute polarizing properties of the sample, which can then be retinal [109, 110] (see Fig. 4) or choroidal thickness [112]. mapped onto a depth-resolved OCT intensity image. Miura et al. showed that PS-OCT is complementary to Pircher et al. first noted that light reflected from the other melanin imaging techniques by combining PS-OCT RPE/Bruch’s membrane complex has a highly variable with polarization-sensitive SLO and NIR-AF to study RPE polarization when measured with PS-OCT in vivo in a cells migration in patients with AMD [113]. PS-OCT has Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 8 of 13

Fig. 4 Segmenting the RPE and calculating retinal thicknesses using polarization-sensitive optical coherence tomography (PS-OCT): (a) OCT cross- sectional image of the retina, (b) degree of polarization uniformity (DOPU) image where the RPE has a low DOPU signal (green) compared to the rest of the retina, (c) Segmentation of the RPE based on low DOPU values, (d) position of the inner limiting membrane (blue) and RPE (red), (e) en face average intensity OCT image of the fundus, (f) corresponding retinal thickness calculated as the distance between the inner limiting membrane and the RPE. Reprinted from [109], under creative commons license

also been performed in combination with other functional Bothphenomenacauseachangeinopticalpathlength, OCT modalities, such as OCT angiography, to acquire which is detected as a change in the OCT phase signal. information not only about the RPE but also about the The PT-OCT signal intensity is proportional to the ab- structure and vasculature of eyes affected by AMD sorption coefficient of the tissue, which allows for [111, 114, 115]. New algorithms [116]andinstruments quantitative measurements of the contrast agent con- [117] have also been developed for PS-OCT to improve centration [119]. PT-OCT was first used to detect mel- the detection of melanin and improve axial resolution anin by Makita et al. to image cutaneous melanin with down to < 1 μm. PT-OCT [120]. PT-OCT was first performed in the eye Photothermal OCT (PT-OCT) is another type of func- by Lapierre-Landry et al. where signal from melanin was tional OCT technique [118, 119]. PT-OCT detects optical detected in the RPE in pigmented mice but absent in albino absorbers in tissues, with similar resolution and imaging mice [121]. A follow-up study was performed in depth as OCT. PT-OCT takes advantage of the photother- tyrosinase-mosaiczebrafish,ageneticlineinwhichthe mal effect, where photons absorbed by the contrast agent zebrafish have pigmented and non-pigmented regions (e.g. melanin) are re-emitted as heat. To perform PT-OCT, within the RPE of each eye. This study confirmed that the an amplitude-modulated laser is combined to a phase-sen- PT-OCT signal is specific to melanin in the zebrafish eye sitive OCT system, with the wavelength of this additional [122]. PT-OCT also detected melanosome migration within laser corresponding to the absorption peak of the contrast the RPE by comparing dark-adapted and light-adapted agent. The increase in temperature following photon ab- wild-type zebrafish (see Fig. 5)[122]. sorption causes a thermoelastic expansion surrounding the Both PS-OCT and PT-OCT are considered functional absorber, and a change in the refractive index of the tissue. OCT techniques. They produce high-resolution images Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 9 of 13

Fig. 5 Melanosome migration in the zebrafish RPE due to light- and dark-adaptation as seen with photothermal optical coherence tomography (PT-OCT). a-b OCT cross-sectional images of the zebrafish retina with (c-d) co-registered PT-OCT images showing melanin distribution due to light- or dark-adaptation of the zebrafish, with (e-f) corresponding histology sections. White arrowheads indicate different structures where melanin is present [co-registered between images (a) and (c), and (b) and (d), approximate location for images (e) and (f)]. Scale bar: 50 μm for OCT and PT-OCT images, 25 μm for histology. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptors; ISe, photoreceptor inner segment ellipsoid zone; OS, photoreceptor outer segment. Reprinted from [122] under creative commons license like OCT and they both can acquire volumetric images Conclusion of the retina that are perfectly co-registered to the OCT Melanin is present in the iris, choroid, and RPE, and intensity images. Both PS-OCT and PT-OCT instru- may act as a protector to the photoreceptors to promote ments can be combined to other modalities such as the overall health of the retina. Changes in pigmentation OCT angiography to perform multimodal imaging. As are observed in diseases such as albinism, retinitis pigment- PS-OCT and PT-OCT use different contrast mecha- osa and AMD, and studying these pigmentation changes nisms to detect melanin (polarization-scrambling and could offer insights on disease mechanism, disease progres- absorption, respectively), they can provide complemen- sion and treatment options. Here we reviewed non-invasive tary information about melanin distribution within the techniques to detect and quantify retinal melanin in the liv- retina. PS-OCT has the advantage of being low in illu- ing eye. These methods have advantages over traditionally mination power, and it has been performed in both ani- used ex vivo methods, since they can be used for longitu- mal models and patients with a range of eye conditions. dinal studies in animal models, where cost, time, labor and It has the potential of being a quantitative imaging mo- inter-animal variability are reduced by imaging the same dality for melanin, although it is unclear how the signal animal over many time points. Many non-invasive imaging is dependent on the shape and size of the melanin gran- methods can also be used in patients for diagnosis and ules and how small changes in pigmentations would be treatment, which is not possible with ex vivo methods. detected. PT-OCT has a more straightforward relation- In this review, we covered multiple techniques that ship with the absorption coefficient of a sample, with a have been used to detect melanin using a variety of con- linear increase in PT-OCT signal as a function of ab- trast mechanisms. Changes in pigmentation can be seen sorption. The PT-OCT signal is thus highly sensitive to using fundus photography, but observations are only small changes in pigmentation within the RPE. However, qualitative and the signal produced by melanin contained PT-OCT has yet to be performed in the human eye, and in the RPE cannot be separated from the signal produced laser powers within safe levels (below ANSI standards) in the choroid. Fundus reflectometry can quantify melanin have only been demonstrated ex vivo [123]. In conclu- in the RPE, but the complex models required for quantifi- sion, both PS-OCT and PT-OCT have a high axial reso- cation make this technology difficult to implement in lution and can separate the RPE from the choroid, but practice. NIR-AF can be accomplished using commercially while PS-OCT has been used to study multiple diseases available SLO instruments and produces images that are in both animal models and patients, PT-OCT has only simple to interpret by a clinician. However, it is difficult to been recently demonstrated in the eye in animal models. quantify melanin across multiple eyes using NIR-AF and Lapierre-Landry et al. Journal of Biological Engineering (2018) 12:29 Page 10 of 13

RPE melanin cannot be separated from choroidal melanin Publisher’sNote with the existing axial sectioning capabilities of commer- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. cial SLOs. PA imaging uses an ultrasound transducer to produce three-dimensional images of the eye and a pulsed Author details 1 2 laser to detect optical absorbers such as melanin. The PA Morgridge Institute for Research, Madison, WI, USA. Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA. signal intensity is directly correlated with melanin absorp- 3Department of Cell Biology, Neurobiology & Anatomy, Medical College of tion and recent advances have made it possible to separate Wisconsin, Milwaukee, WI, USA. 4Department of Ophthalmology & Visual 5 the signal from RPE and the choroid. However, the axial Sciences, Medical College of Wisconsin, Milwaukee, WI, USA. Department of Biomedical Engineering, University of Wisconsin Madison, Madison, WI, USA. resolution is still limited, and the technique has not been 6Department of Pediatrics, Case Western Reserve University, Cleveland, OH, performed the human eye. Finally, OCT is a three-dimen- USA. sional imaging technique that is commonly used in the Received: 27 September 2018 Accepted: 22 November 2018 clinic. Since melanin does not produce a specific change in OCT signal, functional OCT techniques such as PS-OCT and PT-OCT have been developed to detect References melanin using its polarization-scrambling properties and 1. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. its absorption properties, respectively. While PS-OCT has 2005;85(3):845–81. 2. Sarna T. New trends in photobiology: properties and function of the ocular been used in multiple animal models and in patients, melanin—a photobiophysical view. J Photochem Photobiol B. 1992;12(3):215–58. 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